Type I Interferon Transcriptional Signature in Neutrophils and Low-Density Granulocytes Are Associated with Tissue Damage in Malaria

Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. However, the role of these polymorphonuclear cells in mediating either the resistance or the pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, enhanced release of reactive oxygen species and myeloperoxidase, and a high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed intense recruitment of neutrophils to liver sinusoids. Neutrophil migration and IL-1beta and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggest that type I interferon signaling has a central role in neutrophil activation and malaria pathogenesis

The CD14+CD16+ inflammatory monocyte subset displays increased mitochondrial activity and effector function during acute Plasmodium vivax malaria

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax-infected patients display significant increase in circulating monocytes, which were defined as CD14(+)CD16- (classical), CD14(+)CD16(+) (inflammatory), and CD14loCD16(+) (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16(+) cells, in particular the CD14(+)CD16(+) monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14(+) were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14(+)CD16(+) monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-alpha and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14(+)CD16(+) cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection

Mycobacterial antigen driven activation of CD14++ CD16-monocytes is a predictor of tuberculosis-associated immune reconstitution inflammatory syndrome

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14++CD16− monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment

Disrupted iron metabolism and mortality during co-infection with malaria and an intestinal gram-negative extracellular pathogen

Individuals with malaria exhibit increased morbidity and mortality when infected with Gram-negative (Gr−) bacteria. To explore this experimentally, we performed co-infection of mice with Plasmodium chabaudi and Citrobacter rodentium, an extracellular Gr− bacterial pathogen that infects the large intestine. While single infections are controlled effectively, co-infection results in enhanced virulence that is characterized by prolonged systemic bacterial persistence and high mortality. Mortality in co-infected mice is associated with disrupted iron metabolism, elevated levels of plasma heme, and increased mitochondrial reactive oxygen species (ROS) production by phagocytes. In addition, iron acquisition by the bacterium plays a key role in pathogenesis because co-infection with a mutant C. rodentium strain lacking a critical iron acquisition pathway does not cause mortality. These results indicate that disrupted iron metabolism may drive mortality during co-infection with C. rodentium and P. chabaudi by both altering host immune responses and facilitating bacterial persistence

Reticulocyte count: comparison among methods

Introduction: The automated counting of reticulocytes has some advantages over the manual method routinely used in clinical laboratories. Technological innovations provide more statistically reliable results, while optimizing the time to perform this test. However, the cost for implementing the automated procedure in laboratory routines still constitutes a barrier to its use in small- and medium-size Brazilian laboratories. Objective: This study evaluated the performance of a new laboratory protocol for reticulocyte counting by flow cytometry using acridine orange (FC/AO), compared with the manual method and with another automated one by flow cytometry using the commercial kit BD Retic-Count (FC/RC) Conclusion: The results showed that, besides being comparable to the manual method, still considered standard, the evaluated new protocol is economically more advantageous than the automated methods currently available, and its cost is comparable to that of the manual method for laboratories that already have appropriate equipment and infrastructure

Dynamics of T-Lymphocyte Activation Related to Paradoxical Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome in Persons With Advanced HIV

Most persons living with HIV (PLWH) experience a significant restoration of their immunity associated with successful inhibition of viral replication after antiretroviral therapy (ART) initiation. Nevertheless, with the robust quantitative and qualitative restoration of CD4(+) T-lymphocytes, a fraction of patients co-infected with tuberculosis develop immune reconstitution inflammatory syndrome (TB-IRIS), a dysregulated inflammatory response that can be associated with significant tissue damage. Several studies underscored the role of adaptive immune cells in IRIS pathogenesis, but to what degree T lymphocyte activation contributes to TB-IRIS development remains largely elusive. Here, we sought to dissect the phenotypic landscape of T lymphocyte activation in PLWH coinfected with TB inititating ART, focusing on characterization of the profiles linked to development of TB-IRIS. We confirmed previous observations demonstrating that TB-IRIS individuals display pronounced CD4(+) lymphopenia prior to ART initiation. Additionally, we found an ART-induced increase in T lymphocyte activation, proliferation and cytotoxicity among TB-IRIS patients. Importantly, we demonstrate that TB-IRIS subjects display higher frequencies of cytotoxic CD8(+) T lymphocytes which is not affected by ART. Moreover, These patients exhibit higher levels of activated (HLA-DR(+)) and profilerative (Ki-67(+)) CD4(+) T cells after ART commencenment than their Non-IRIS counterparts. Our network analysis reveal significant negative correlations between Total CD4(+) T cells counts and the frequencies of Cytotoxic CD8(+) T cells in our study population which could suggest the existance of compensatory mechanisms for Mtb-infected cells elimination in the face of severe CD4(+) T cell lymphopenia. We also investigated the correlation between T lymphocyte activation profiles and the abundance of several inflammatory molecules in plasma. We applied unsupervised machine learning techniques to predict and diagnose TB-IRIS before and during ART. Our analyses suggest that CD4(+) T cell activation markers are good TB-IRIS predictors, whereas the combination of CD4(+) and CD8(+) T cells markers are better at diagnosing TB-IRIS patients during IRIS events Overall, our findings contribute to a more refined understanding of immunological mechanisms in TB-IRIS pathogenesis that may assist in new diagnostic tools and more targeted patient management

Neutrophil Paralysis in Plasmodium vivax Malaria

Plasmodium vivax is responsible for approximately 60–80% of the malaria cases in the world, and contributes to significant social and economic instability in the developing countries of Latin America and Asia. The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. The innate immune receptors recognize Plasmodium sp. and initiate a broad spectrum of host defense mechanisms that mediate resistance to infection. However, the innate immune response is the classic “two-edged sword”, and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α during acute malaria. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Unexpectedly, we noticed an impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. We proposed that neutrophil paralysis is in part responsible for the enhanced susceptibility to bacterial infection observed in malaria patients

Analysis of the immunological biomarker profile during acute zika virus infection reveals the overexpression of CXCL10, a chemokine linked to neuronal damage

BACKGROUND Infection with Zika virus (ZIKV) manifests in a broad spectrum of disease ranging from mild illness to severe neurological complications and little is known about Zika immunopathogenesis. OBJECTIVES To define the immunologic biomarkers that correlate with acute ZIKV infection. METHODS We characterized the levels of circulating cytokines, chemokines, and growth factors in 54 infected patients of both genders at five different time points after symptom onset using microbeads multiplex immunoassay; comparison to 100 age-matched controls was performed for statistical analysis and data mining. FINDINGS ZIKV-infected patients present a striking systemic inflammatory response with high levels of pro-inflammatory mediators. Despite the strong inflammatory pattern, IL-1Ra and IL-4 are also induced during the acute infection. Interestingly, the inflammatory cytokines IL-1β, IL-13, IL-17, TNF-α, and IFN-γ; chemokines CXCL8, CCL2, CCL5; and the growth factor G-CSF, displayed a bimodal distribution accompanying viremia. While this is the first manuscript to document bimodal distributions of viremia in ZIKV infection, this has been documented in other viral infections, with a primary viremia peak during mild systemic disease and a secondary peak associated with distribution of the virus to organs and tissues. MAIN CONCLUSIONS Biomarker network analysis demonstrated distinct dynamics in concurrence with the bimodal viremia profiles at different time points during ZIKV infection. Such a robust cytokine and chemokine response has been associated with blood-brain barrier permeability and neuroinvasiveness in other flaviviral infections. High-dimensional data analysis further identified CXCL10, a chemokine involved in foetal neuron apoptosis and Guillain-Barré syndrome, as the most promising biomarker of acute ZIKV infection for potential clinical application. © 2018, Fundacao Oswaldo Cruz. All rights reserved

Differential Use of TLR2 and TLR9 in the Regulation of Immune Responses during the Infection with Trypanosoma cruzi

Submitted by Nuzia Santos ([email protected]) on 2018-12-05T18:14:36Z No. of bitstreams: 1 Differential Use of TLR2 and TLR9 .pdf: 5776313 bytes, checksum: 3206b5a0f2e440a9c95d8318a58fc1f8 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2018-12-05T18:20:21Z (GMT) No. of bitstreams: 1 Differential Use of TLR2 and TLR9 .pdf: 5776313 bytes, checksum: 3206b5a0f2e440a9c95d8318a58fc1f8 (MD5)Made available in DSpace on 2018-12-05T18:20:21Z (GMT). No. of bitstreams: 1 Differential Use of TLR2 and TLR9 .pdf: 5776313 bytes, checksum: 3206b5a0f2e440a9c95d8318a58fc1f8 (MD5) Previous issue date: 2013Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil/Division of Infectious Diseases and Immunology. University of Massachusetts Medical School. Worcester, MA, Untied States of America.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilPathogens express ligands for several TLRs that may play a role in the induction or control of the inflammatory response during infection. Concerning Trypanosoma cruzi, the agent of Chagas disease, we have previously characterized glycosylphosphatidylinositol (GPI) anchored mucin-like glycoproteins (tGPI-mucin) and unmethylated CpG DNA sequences as TLR2 and TLR9 agonists, respectively. Here we sought to determine how these TLRs may modulate the inflammatory response in the following cell populations: F4/80+CD11b+ (macrophages), F4/80lowCD11b+ (monocytes) and MHCII+CD11chigh (dendritic cells). For this purpose, TLR2−/− and TLR9−/− mice were infected with Y strain of T. cruzi and different immunological parameters were evaluated. According to our previous data, a crucial role of TLR9 was evidenced in the establishment of Th1 response, whereas TLR2 appeared to act as immunoregulator in the early stage of infection. More precisely, we demonstrated here that TLR2 was mainly used by F4/80+CD11b+ cells for the production of TNF-α. In the absence of TLR2, an increased production of IL-12/IL-23p40 and IFN-γ was noted suggesting that TLR2 negatively controls the Th1 response. In contrast, TLR9 was committed to IL-12/IL-23p40 production by MHCII+CD11chigh cells that constitute the main source of IL-12/IL-23p40 during infection. Importantly, a down-regulation of TLR9 response was observed in F4/80+CD11b+ and F4/80lowCD11b+ populations that correlated with the decreased TLR9 expression level in these cells. Interestingly, these cells recovered their capacity to respond to TLR9 agonist when MHCII+CD11chigh cells were impeded from producing IL-12/IL-23p40, thereby indicating possible cross-talk between these populations. The differential use of TLR2 and TLR9 by the immune cells during the acute phase of the infection explains why TLR9- but not TLR2-deficient mice are susceptible to T. cruzi infection

Comparison of the capacity of F4/80⁺CD11b⁺, F4/80^lowCD11b⁺ and MHCII⁺CD11c^high populations to respond to TLR9 agonist during acute phase of <i>T.cruzi</i> infection.

<p>Intracellular TNF-α and IL-12/IL-23p40 were analyzed by flow cytometry in spleen from C57BL/6 mice seven days post-infection. Splenic cells were cultured in medium alone, with CpG DNA (1 µg/ml), LPS (1 µg/ml) or Pam3Cys (1 µg/ml). (A) Frequencies of splenic TNF-α⁺ or IL-12/IL-23p40⁺ cells (F4/80⁺CD11b⁺, F4/80^lowCD11b⁺ and MHCII⁺CD11c^high) after stimulation with CpG DNA (1 µg/ml) (mean ± SD of four mice) or (B) frequencies of splenic TNF-α⁺ or IL-12/IL-23p40⁺ cells (F4/80⁺CD11b⁺ and F4/80^lowCD11b⁺) stimulated with LPS (1 µg/ml) or Pam3Cys (1 µg/ml) (mean ± SD of four mice) isolated from infected and non-infected mice. Data are representative of three independent experiments. *p<0.05 and **p<0.01 indicate statistical significance when comparing the percentage of the same cell population from infected or not infected mice cultured in the same conditions.</p